The development and evolution of animal forms and patterns is a central topic in biology. The long-term goal of the proposed research is to understand how developmental genes that are conserved between all animals, a.k.a. toolkit genes, become co-opted into new gene-regulatory networks, using the polka-dotted species Drosophila guttifera as a model. The objective of this application is to identify the toolkit genes that control the complex pigmentation pattern on the D. guttifera abdomen and to identify the cis-regulatory elements (CREs) of the downstream target genes yellow (y) and tan (t), which are terminal pigmentation genes that are co- expressed in identical complex patterns. The rationale underlying the proposed research is that very little is known about how complex animal color patterns are generated and how transcription factors co-regulate downstream target genes in identical expression patterns. The central hypothesis is that morphogens and homeobox transcription factors co-regulate the two terminal pigmentation genes y and t to produce a complex abdominal melanin pattern in D. guttifera. The central hypothesis will be tested by pursuing the following specific aims: 1) Identify the developmental genes/pathways that regulate the pigmentation genes y and t, 2) Characterize the cis-regulatory elements of y and t that respond to the developmental pathways, and 3) Per- form functional necessity and sufficiency tests for selected toolkit genes. The candidate gene approaches using in situ hybridization and immunohistochemistry on developing abdomens will be employed for the study proposed under Aim 1. The goal is to find the genes that play a role in inducing the complex abdominal color pattern, e.g. genes that are expressed in patterns foreshadowing the adult pigmentation pattern. The goal of the research proposed under Aim 2 is to investigate how toolkit genes become co-opted into new gene- regulatory networks. This will be accomplished by fluorescent reporter gene assays in transgenic D. guttifera to reveal the location, size, and transcription factor binding sites of the CREs that drive the complex y and t expression patterns on the developing abdomen. The goal of Aim 3 is to produce Gal4 driver lines and use the Gal4-UAS system to ectopically express toolkit gene cDNA and RNAi constructs in D. guttifera. Using this genetic tool, manipulating the abdominal color pattern will provide direct genetic evidence for the involvement of the candidate genes in either activating or repressing the pattern. The contribution will be significant because the candidate toolkit genes that foreshadow the pigmentation pattern in the D. guttifera abdomen are related to disease and disability-causing genes in humans. It is thus important to gain a deeper understanding of how toolkit genes become co-opted into gene-regulatory networks and how they co-regulate downstream target genes. The proposed research project is innovative because it employs the emerging model organism D. guttifera, an organism that is sequenced, transgenically modifiable, and that displays complex color patterns, which can be used as convenient markers that indicate the activity of toolkit gene pathways in an insect model.